The present disclosure relates to the biosynthetic production of 5-amino-5-deoxyshikimic acid or kanosamine and their conversion to oseltamivir carboxylates and other derivatives.
Aminoshikimate is an industrially important compound that can be used as a starting material in the formation of oseltamivir carboxylates for use in producing the antiviral drug formulation, TAMIFLU (Hoffmann-La Roche). Oseltamivir carboxylates are commercially produced using shikimic acid as a starting material. Traditionally, the shikimic acid has been isolated from plants, especially Illicium species, which include Chinese star anise (I. verum) and Japanese star anise (I. anisatum). Star anise seeds are industrially processed in a ten-stage procedure, which takes about a year, in order to obtain shikimic acid. A shortage of shikimic acid, sometimes attributed to insufficient quantities of star anise, has been cited as a potential impediment to the production of oseltamivir carboxylates. As a result, recombinant microbes, engineered to exhibit increased shikimic acid production, have been used to produce shikimic acid to help meet this need.
However, even with microbial synthesis of shikimic acid, the cost of converting shikimic acid to oseltamivir carboxylates has remained relatively constant. Two different major chemosynthetic routes have been reported for the conversion to oseltamivir phosphate, which is the active ingredient present in TAMIFLU, each of which utilizes many steps, e.g.: 10 steps, including three explosive and/or toxic azide derivatives; or 17 steps in an azide-free process. In both of these routes, about 4 of the steps are performed in order to add an amino group substituent at the 5-position of the shikimate ring. See, e.g., C. U. Kim et al., J. Am. Chem. Soc. 119(4):681-90 (Jan. 29, 1997); J. C. Rohloff et al., J. Org. Chem. 63(13):4545-50 (Jun. 26, 1998); M. Karpf & R. Trussardi, J. Org. Chem. 66(6):2044-51 (Mar. 23, 2001); S. Abrecht et al., Chimia 58(9):621-29 (2004); Y.-Y. Yeung et al., J. Am. Chem. Soc. 128(19):6310-311 (May 17, 2006); Y. Fukuta et al., J. Am. Chem. Soc. 128(19):6312-13 (May 17, 2006); and T. Mita et al., Org. Lett. 9(2):259-62 (Jan. 18, 2007).
As a result, providing a process that does not require those steps can significantly improve both the speed and economics of the production of oseltamivir phosphate or other oseltamivir carboxylates. One way to help achieve this goal could be to provide biosynthetic 5-aminoshikimic acid, i.e. 5-amino-5-deoxyshikimic acid, as a starting material for the chemosynthetic oseltamivir phosphate production process.
Two biosynthetic routes for production of aminoshikimic acid have been reported. In the first, the wild-type bacterium, Amycolatopsis mediterranei (ATCC 21789), has been found capable of anabolic synthesis of aminoshikimate from glucose, using a biosynthetic route that involves formation of the high energy intermediate, UDP-glucose, transformation to UDP-kanosamine and then to kanosamine, followed by conversion of the kanosamine, in multiple steps, to aminoshikimate.
In the second route, two different organisms are used: 1) Bacillus pumilus (ATCC 21143), used for anabolic synthesis of kanosamine, also via the high energy UDP-glucose pathway; and 2) a recombinant E. coli, used to convert the resulting kanosamine to aminoshikimate. J. Guo & J. Frost, Org. Lett. 6(10):1585-88 (May 13, 2004) (published online Apr. 14, 2004 as DOI 10.1021/ol049666e); J. Guo & J. Frost, J. Am. Chem. Soc. 124(36):10642-43 (Sep. 11, 2002); also J. Guo & J. Frost, J. Am. Chem. Soc. 124(4):528-29 (Jan. 30, 2002). Yet, for commercial applications, this process would require two separate fermentations, with an intervening recovery of the kanosamine intermediate so as to at least partially remove Bacillus-expressed toxins and antimicrobial peptides therefrom. These multiple steps would introduce significant expense and decreased yields of aminoshikimate by loss of kanosamine.
Moreover, because both of these are high energy processes, they are metabolically expensive, and use of these processes would present commercially expensive routes to obtain an aminoshikimate starting material for oseltamivir phosphate production. A less energy-intensive process would be important in order to obtain an economically advantageous route.
As a result, it would be beneficial to provide a more efficient, less expensive route for biosynthesis of aminoshikimate. It would likewise be beneficial to provide an overall process for production of oseltamivir carboxylates that is similarly more efficient and less expensive than the current process. It would also be desirable to provide a process that can be used to produce other useful intermediates, as well.